Resolution of the Hypoxylon fuscum Complex (Hypoxylaceae, Xylariales) and Discovery and Biological Characterization of Two of Its Prominent Secondary Metabolites.

Hypoxylon, a large, cosmopolitan genus of Ascomycota is in the focus of our current poly-thetic taxonomic studies, and served as an excellent source for bioactive secondary metabolites at the same time. The present work concerns a survey of the Hypoxylon fuscum species complex based on specimens from Iran and Europe by morphological studies and high performance liquid chromatography coupled to mass spectrometry and diode array detection (HPLC-MS-DAD). Apart from known chemotaxonomic markers like binaphthalene tetrol (BNT) and daldinin F, two unprece-dented molecules were detected and subsequently isolated to purity by semi preparative HPLC. Their structures were established by nuclear-magnetic resonance (NMR) spectroscopy as 3'-malonyl-daldinin F ( 6 ) and pseudofuscochalasin A ( 4 ). The new daldinin derivative 6 showed weak cytotoxicity towards mammalian cells but bactericidal activity. The new cytochalasin 4 was compared to cytochalasin C in an actin disruption assay using fluorescence microscopy of human osteo-sarcoma U2OS cells, revealing comparable activity towards F-actin but being irreversible compared to cytochalasin C. Concurrently, a multilocus molecular phylogeny based on ribosomal and proteinogenic nucleotide sequences of Hypoxylon species resulted in a well-supported clade for H. fuscum and its allies. From a comparison of morphological, chemotaxonomic and phylogenetic evidence, we introduce the new species H. eurasiaticum and H. pseudofuscum .